Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3'UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3'UTR; and 5) the most distal AU-rich elements present in HIF-1α 3'UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.